Digital Holographic Microscopy (DHM) is a powerful imaging technique allowing, from a single amplitude image acquisition (hologram), the reconstruction of the entire complex wave front (amplitude and phase), reflected by or transmitted through an object. Because holography is an interferometric technique, the reconstructed phase leads to a sub-wavelength axial accuracy (below λ/100). Nevertheless, this accuracy is difficult to obtain from a single hologram. Indeed, the reconstruction process consisting to process the hologram with a digital reference wave (similar to classical holographic reconstruction) seems to need a-priori knowledge about the physical values of the setup. Furthermore, the introduction of a microscope objective (MO), used to improve the lateral resolution, introduces a wave front curvature in the object wave front. Finally, the optics of the set-up can introduce different aberrations that decrease the quality and the accuracy of the phase images. We propose here an automatic procedure allowing the adjustment of the physical values and the compensation for the phase aberrations. The method is based on the extraction of reconstructed phase values, along line profiles, located on or around the sample, in assumed to be flat area, and which serve as reference surfaces. The phase reconstruction parameters are then automatically adjusted by applying curve-fitting procedures on the extracted phase profiles. An example of a mirror and a USAF test target recorded with high order aberrations (introduced by a thick tilted plate placed in the set-up) shows that our procedure reduces the phase standard deviation from 45 degrees to 5 degrees.
Digital Holographic Microscopy (DHM) provides three-dimensional (3D) images with a high vertical accuracy in the
nanometer range and a diffracted limited transverse resolution. This paper focuses on 3 different tomographic applications
based on DHM. First, we show that DHM can be combined with time gating: a series of holograms is acquired at different
depths by varying the reference path length, providing after reconstruction images of slices at different depths in the
specimen thanks to the short coherence length of the light source. Studies on enucleated porcine eyes will be presented.
Secondly, we present a tomography based on the addition of several reconstructed wavefronts measured with DHM at
different wavelengths. Each wavefront phase is individually adjusted to be equal in a given plane of interest, resulting in a
constructive addition of complex waves in the selected plane and destructive addition in the others. Varying the plane of
interest enables the scan of the object in depth. Thirdly, DHM is applied to perform optical diffraction tomography of a
pollen grain: transmission phase images are acquired for different orientations of the rotating sample, then the 3D
refractive index spatial distribution is computed by inverse radon transform. The presented works will exemplify the
versatility of DHM, but above all its capability of providing quantitative tomographic data of biological specimen in a
quick, reliable and non-invasive way.
We report on a method called Digital Holographic Microscopy (DHM) for the numerical reconstruction of digital holograms taken with a microscope. It allows for simultaneous amplitude and quantitative phase contrast imaging. The reconstruction method computes the propagation of the complex optical wavefront diffracted by the object and is used to determine the refractive index and/or shape of the object with an accuracy in the nanometer range along the optical axis. A single hologram is needed for reconstruction. The method requires the adjustment of several reconstruction parameters. The adjustment is performed automatically by using a suitable algorithm. The method has been applied to the measurement of several integrated optics devices, MOEMS, and integrated micro-optical components: microlenses.
Few models, based on the diffraction theory, are proposed in order to evaluate the point spread function of different microscope objectives used in a digital holographic microscope. Because in holography the phase information is essential, a 3D amplitude point spread function (APSF), modulus and phase, is necessary, in order to properly deconvolute the 3D images obtained. Scalar Debye theory, paraxial approximation and vectorial Debye theory are used to solve the diffraction problem and the theoretical predicted 3D APSFs obtained with these models are compared.
A simple model is proposed in order to evaluate the complex amplitude point spread function (intensity and phase) of a microscope objective. The model is based on the Fresnel diffraction theory and takes also into account the possible optical aberrations. Experimental evaluation of this amplitude point spread function has been carried out by using a holographic set-up and 60 nanometers gold spheres as punctual objects. The measured values are then compared with the theoretical predicted model.
The aim of Optical Digital Holography, applied to cells and tissue imaging, is to provide an accurate 3D imaging of biologic materials, down to the microscopic scale. The method has been developed to yield a very precise determination of cells and tissues morphology. Targeted accuracies are in the sub-micron range and allow for the observation of very small movements and deformations, produced, in particular, by depolarization of excitable cells and their metabolic activities. Direct imaging of tissue structures by the newly developed digital holography is deemed to offer unique investigation means in biology and medicine and attractive diagnostic capabilities.
We report on new developments of a technique called Digital Holographic Microscopy (DHM), for the numerical reconstruction of digital holograms taken in microscopy, which allows simultaneous amplitude and quantitative phase contrast imaging. The reconstruction method computes the propagation of the complex optical wavefront diffracted by the object and is used to determine the refractive index and/or shape of the object with accuracies in the nanometer range along the optical axis.. The method requires the acquisition of a single hologram. The technique comprises the recording of a digital hologram of the specimen by means of a standard CCD camera at the exit of a Mach-Zehnder or Michelson type interferometer. The quantitative nature of the reconstructed phase distribution has been demonstrated by an application to surface profilometry where step height differences of a few nanometers have been measured. Another application takes place in biology for transmission phase- contrast imaging of living cells in culture. The resolution for thickness measurements depends on the refractive index of the specimen and a resolution of approximately 30 nanometers in height, and about half of a micro in width, has been achieved for living neural cells in cultures by using a high numerical aperture.
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